Chlamydia trachomatis is the leading cause of preventable blindness and a major cause of sexually transmitted disease. A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood. We have preformed a functional genomic anaylsis on C. trachomatis through a combinated analysis of comparative genomics, pathogenesis, and basic biological properties of the pathogen. We have found that virulence determinants map to a polymorphic region of the otherwise highly conversed genomes termed the plasticity zone (PZ). We have identified two new virulence determinants in the PZ, a chlamydial cytotoxin and genes encoding the tryptophan repressor and alpha and beta subunits of tryptophan synthase. We present findings indicating that these two gene families function in mucosal tropism properties of the pathogen (cytotoxin positive strains) and in the evasion of host defenses (tryptophan synthase positive strains). It has been proposed that chlamydiae have cytotoxic activity that contributes to this pathology, but a toxin has not been identified. We have identified a cytotoxin associated with certain chlamydial strains and believe that the toxin represents a novel virulence factor that is important in the pathophysiology of chlamydial diseases. C. trachomatis isolates are separated into biovariants depending on the disease they cause. The trachoma biovars produce localized infection of the eye or genital mucosa. Infections with these strains are a major cause of STD and are characterized by persistent infection of mucosal surfaces resulting in chronic inflammatory disease. In women these infections are manifested as pelvic inflammatory disease whose sequelae can produce tubal blockage and infertility as well as tubal pregnancy. In contrast, the non-trachoma strains cause lymphogranuloma venereum (LGV) an invasive STD. LGV strains transiently infect the genital mucosa but then rapidly disseminate to the draining lymph nodes where the parasite infects monocytes and granuloma formation. An understanding of the molecular basis for these marked differences in virulence and pathogenesis are likely key to the identification of new parasite molecules that can be targeted for novel intervention strategies against chlamydial disease. The genomes of a trachoma-STD and a LGV biovariant have recently been sequenced and annotated. Comparative genomic analysis showed remarkable similarity of the small1-1.2 million base pair genomes of each biovariant despite their marked differences in tissue tropism and disease manifestation. However, a single gene family (CT 166-169) with homology to the large clostridial cytotoxin toxin B was found in the trachoma-STD biovariants genome which was not present in the LGV genome. It is therefore possible that this putative chlamydial toxin plays a critical role in the virulence and pathogenesis of the trachoma-STD biovariants. We have therefore focused this project on studies to define whether the trachoma-STD variants express a biologically active toxin and if so what role the toxin plays in the pathogenesis of disease caused by these medically important pathogens. Our first goal was to document that trachoma-STD biovariants and not LGV strains express an active toxin. We found that inoculation of HeLa 229 cells with a high multiplicity of infection of a trachoma-STD strain makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B. Cytotoxin gene transcripts were detected in chlamydial-infected cells and a protein with expected molecular mass was present in infected cells during the period of cytotoxicity. The protein was present transiently infected cells during the period of cytotoxicity. Together, these results provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary bodies and is delivered to host cells very early during infection. We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C. trachomatis disease. The conservation of trpRBA is particularly striking because the remaining genes of the tryptophan operon are absent leaving no biochemical connection for retaining the capability to both regulate and make the synthase. IFN-gamma controls chlamydial growth by decreasing the availability of L-tryptophan in host cells; a mechanism that has been implicated in both eradication and persistence. We show that chlamydial strains that colonize the genital mucosae, but not the ocular mucosae, contain a functional tryptophan synthase. Interestingly, the genital strains alpha-polypeptide has acquired mutations that map to the enzymes active site that binds the normal substrate indole glycerol phosphate (IGP). We demonstrate by genetic complementation that the chlamydial tryptophan synthase is unable to utilize IGP but can utilize exogenous indole for the synthesis of tryptophan. In HeLa cells, the potent chlamydial inhibitory effect of IFN-gamma was completely reversed by exogenous indole. These findings suggest a strong selection for C. trachomatis genital stains to control the synthesis of tryptophan and imply that a unique molecular relationship exists between genital tropic strains and the host. We hypothesize that in vivo co-infecting vaginal flora might be a source of indole. Thus, trpRBA could represent a new C. trachomatis virulence factor important in the pathogenesis of chlamydial genital infection. It may function by utilizing microbially produced indole as a substrate to produce tryptophan; thereby evading the effect of IFN-gamma mediated host defense and allowing the establishment of persistent infection.